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Global carbon storage in soils is widely discussed today because of climate uncertainty and maintaining sustainable agricultural production. Human intervention in agricultural or energy production poses many changes in soil management, which highly affects soil quality/health. Permanent grasslands fulfil a wide range of ecosystem functions and have a high potential for increasing arable land. Today, grasslands are becoming more and more intensively used, fertilized and disturbed. Optimizing their management is essential to maintain a resilient and stable ecosystem. The produced biomass is used as a forage or for energy production. We aimed at the impact of long-term grassland liming on the total content of soil organic carbon (SOC), humic substances (CHS), and microbial biomass (Cmic). Furthermore, soil reaction and available nutrient content were evaluated. Soil samples were collected from a split-plot field experiment at Mendel University in Brno (locality Kameničky). The soil was classified as Dystric Planosol Siltic, medium textured, strongly acidic, with high soil organic carbon content. The yearly liming rate was 1.4 t/ha CaO. The linkage between the soil pH, SOC, Cmic, and available nutrient content was evaluated by the multivariate exploratory techniques and regression models. Results showed that long-term liming affects both soil biota and carbon storage. [ABSTRACT FROM AUTHOR]